US2023242916A1PendingUtilityA1
Method and drug for treating hurler syndrome
Assignee: EDIGENE THERAPEUTICS BEIJING INCPriority: Apr 15, 2020Filed: Apr 15, 2021Published: Aug 3, 2023
Est. expiryApr 15, 2040(~13.7 yrs left)· nominal 20-yr term from priority
C12N 15/1137A61K 48/0075C12N 2320/34C12N 2310/315A61P 3/00A61K 48/00C12N 15/52C12N 9/2402C12Y 302/01076C12Y 305/04004C12N 9/78C12N 2310/321C12N 2310/344C12N 2740/16043C12N 15/88C12N 15/11C12N 15/113C12N 15/102C12N 2310/31C12N 2310/33C12N 2310/3231
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Claims
Abstract
Disclosed are a LEAPER technique-based nucleic acid drug and a method for treating diseases such as Hurler syndrome by means of using the nucleic acid drug to target and edit RNA. The method comprises: performing an editing from adenosine base to hypoxanthine base on RNA by using the nucleic acid drug to precisely repair the pathogenic G>A mutation site of, for example, Hurler syndrome; thereby recovering the normal in vivo expression of a protein encoded by RNA, such as IDUA.
Claims
exact text as granted — not AI-modified1 . A method for targeted editing of a target RNA in a cell based on the LEAPER technology, wherein the target RNA comprises a transcript sequence of an α-L-iduronidase (IDUA) gene containing a guanosine (G) to adenosine (A) mutation site (target adenosine), and the method comprises:
introducing into the cell an arRNA or a construct comprising the arRNA coding sequence, wherein the arRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and wherein the arRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) by hybridization to the target RNA so as to deaminate the target adenosine in the target RNA.
2 . The method according to claim 1 , wherein the nucleotide (targeting nucleotide) in the arRNA opposite to the target adenosine is a cytidine (C), an adenosine (A) or a uridine (U).
3 . The method according to claim 1 , wherein the target adenosine forms a target triplet together with its 5′ most adjacent nucleotide and 3′ most adjacent nucleotide, and the target triplet is 5′-UAG-3′.
4 . The method according to claim 1 , wherein the 5′ most adjacent nucleotide of the targeting nucleotide is a cytidine (C), a guanosine (G) or a uridine (U).
5 . The method according to claim 1 , wherein the 3′ most adjacent nucleotide of the targeting nucleotide is an adenosine (A).
6 . The method according to claim 1 , wherein the targeting nucleotide forms a targeting triplet together with its 5′ most adjacent nucleotide and 3′ most adjacent nucleotide, and the targeting triplet is 5′-CCA-3′.
7 . The method according to claim 1 , wherein the length of the arRNA is any integer number of nt selected from 61 nt to 121 nt.
8 . The method according to claim 1 , wherein the distance from the targeting nucleotide of the arRNA to the 3′ end of the arRNA is less than the distance from the targeting nucleotide to the 5′ end of the arRNA.
9 . The method according to claim 1 , wherein the distance from the targeting nucleotide of the arRNA to the 3′ end of the arRNA is any integer number of nt selected from 9 nt to 60 nt.
10 . The method according to claim 1 , wherein the distance from the targeting nucleotide of the arRNA to the 5′ end of the arRNA is any integer number of nt selected from 25 nt to 60 nt.
11 . The method according to claim 1 , wherein the arRNA is an arRNA having a length characteristic selected from the group consisting of:
55 nt-c-35 nt, 55 nt-c-25 nt, 55 nt-c-24 nt, 55 nt-c-21 nt, 55 nt-c-20 nt, 55 nt-c-19 nt, 55 nt-c-18 nt, 55 nt-c-17 nt, 55 nt-c-16 nt, 55 nt-c-15 nt, 55 nt-c-14 nt, 55 nt-c-13 nt, 55 nt-c-12 nt, 55 nt-c-11 nt, 55 nt-c-10 nt, 55 nt-c-9 nt, 50 nt-c-20 nt, 50 nt-c-15 nt, and 45 nt-c-20 nt; wherein cytidine is the targeting nucleotide in the arRNA opposite to the target adenosine, and the orientation of the arRNA is 5′ to 3′.
12 . (canceled)
13 . The method according to claim 1 , wherein the arRNA comprises one or more chemical modifications.
14 . The method according to claim 13 , wherein the chemical modification is one or more selected from the group consisting of:
2′-O-methylation modification, phosphorothioation modification in internucleotide linkage, deoxyribonucleotide substitution modification, LNA modification, and 2′-O-(2-methoxyethyl) modification.
15 . The method according to claim 13 , wherein the chemical modification is one or more selected from the group consisting of:
1) 2′-O-methylation modifications in the first 2, 3, 4 or 5 nucleotides; 2) 2′-O-methylation modifications in the last 2, 3, 4, or 5 nucleotides; 3) 2′-O-methylation modifications in all the cytidines except that in the targeting triplet; 4) 2′-O-(2-methoxyethyl) modifications in the first 2, 3 or 4 nucleotides; 5) 2′-O-(2-methoxyethyl) modifications in the last 2, 3 or 4 nucleotides; 6) deoxyribonucleotide substitution modifications of the first 2, 3 or 4 nucleotides; 7) deoxyribonucleotide substitution modifications of the last 2, 3 or 4 nucleotides; 8) phosphorothioation modifications in the first 1, 2, 3, 4 or 5 internucleotide linkages; 9) phosphorothioation modifications in the last 1, 2, 3, 4 or 5 internucleotide linkages; 10) phosphorothioation modifications in all internucleotide linkages except that one or more of the 5′ or 3′ most adjacent internucleotide linkages of each of the nucleotides in the targeting triplet are not modified by phosphorothioation; and (11) phosphorothioation modifications in internucleotide linkages at an interval of 1, 2, 3, or 4 nucleotides except that one or more of the 5′ or 3′ most adjacent internucleotide linkages of each of the nucleotides in the targeting triplet are not modified by phosphorothioation.
16 . (canceled)
17 . The method according to claim 1 , wherein the guanosine (G) to adenosine (A) mutation site targeted by the arRNA is a mutation site in NM_000203.4(IDUA)-c.1205G>A (p.Trp402Ter), or a corresponding mutation site thereof, in the genome of a patient with Hurler syndrome.
18 . The method according to claim 1 , wherein the arRNA comprises any sequence selected from the group consisting of:
1)
(SEQ ID NO: 31)
Gm*Am*Cm-G*Cm-Cm*Cm-A*Cm-Cm*G-Um*
G-Um*G-G*Um-Um*G-Cm*Um-G*Um-Cm*Cm-
A*G-G*A-Cm*G-G*Um-Cm*Cm-Cm*G-G*Cm-
Cm*Um-G*Cm-G*A-Cm*A-Cm*Um-Um*Cm-
G*G-Cm-C-C-A*G-A*G-Cm*Um-G*Cm-Um*
Cm-Cm*Um*Cm*Am;
2)
(SEQ ID NO: 31)
Gm*Am*Cm-G*Cm-Cm*Cm-A*-Cm-Cm*G-
Um*G-Um*G-G*Um-Um*G-Cm*Um-G*Um-
Cm*Cm-A*G-G*A-Cm*G-G*Um-Cm*Cm-Cm*
G-G*Cm-Cm*Um-G*Cm-G*-A-Cm*A-Cm*
Um-Um*Cm-G*G-Cm*Cd-Cd*Ad*-G-A*G-
Cm*Um-G*Cm-Um*Cm-Cm*Um*Cm*Am;
3)
(SEQ ID NO: 31)
Gm*Am*Cm-G*Cm-Cm*Cm-A*Cm-Cm*G-
Um*G-Um*G-G*Um-Um*G-Cm*Um-G*Um-
Cm*Cm-A*G-G*A-Cm*-G-G*Um-Cm*Cm-
Cm*G-G*Cm-Cm*Um-G*-Cm-G*A-Cm*A-
Cm*Um-Um*Cm-G*G-Cm-Cd-Cd*Ad*G-
A*G-Cm*Um-G*Cm-Um*Cm-Cm*Um*
Cm*Am;
4)
(SEQ ID NO: 31)
Gmoe*Amoe*Cmoe*Gmoe-C-C-C-A-C-
C-G-Um-G-Um-G-G-Um-Um-G-C-Um-G-
Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-
G-G-C-C-Um-GC-G-A-C-A-C-Um-Um-
C-G-G-C-C-C-A-G-A-G-C-Um-G-C-
Um-C*Cmoe*Umoe*Cmoe*Amoe;
or
5)
(SEQ ID NO: 31)
G+*A+*C+*G+C-C-C-A-C-C-G-Um-G-
Um-G-G-Um-Um-G-C-Um-G-Um-C-C-
A-G-G-A-C-G-G-Um-C-C-C-G-G-C-
C-Um-GC-G-A-C-A-C-Um-Um-C-G-G-
C-C-C-A-G-A-G-C-Um-G-C-Um-C*
C+*U+*C+*A+;
wherein * indicates a phosphorothioation modification, m indicates a 2′-O-methylation modification, moe indicates a 2′-O-(2-methoxyethyl) modification, + indicates an LNA modification, and d indicates a deoxyribonucleotide substitution modification.
19 . The method according to claim 1 , wherein the arRNA is introduced into the cells by any method selected from the group consisting of: electroporation, liposome transfection, exosome delivery, or lipid-nanoparticle delivery (LNP).
20 . The method according to claim 1 , wherein the amount of arRNA in a single introduction into the cells is 5 to 160 nM.
21 . The method according to claim 1 , wherein several introductions are taken for introducing the arRNA into the cells, and the interval between the adjacent two introductions is ≤21 days.
22 . A method for preventing or treating Hurler syndrome, comprising correcting a pathogenic G>A mutation of Hurler syndrome by using the method according to claim 1 .
23 . An engineered arRNA which is used for targeted editing of a target RNA in a cell based on LEAPER technology, wherein the target RNA comprises a transcript sequence of an α-L-iduronidase (IDUA) gene containing a guanosine (G) to adenosine (A) mutation site (target adenosine), the arRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the arRNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) by hybridization to the target RNA so as to deaminate the target adenosine in the target RNA, the guanosine (G) to adenosine (A) mutation site targeted by the arRNA is NM_000203.4(IDUA)-c.1205G>A (p.Trp402Ter) mutation site, or a corresponding mutation site thereof, in the genome of a patient with Hurler syndrome.
24 - 29 . (canceled)
30 . A construct comprising the coding sequence of the arRNA according to claim 23 .
31 . A composition comprising the arRNA according to claim 23 .Join the waitlist — get patent alerts
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